My co-tutelle Polish-French PhD thesis

Regulatory role of hydrogen cyanide and ROS in dormancy removal of sunflower (Helianthus annuus L.) embryos (Poland-France) - PhD thesis supervised by Christophe Bailly, Francoise Corbineau, Renata Bogatek.

Seed dormancy and germination are very complex phenomena which involve tightly controlled signaling pathways as well as cellular and molecular regulations. In addition to hormones, there are also signaling molecules such as HCN and ROS, which seem to play important roles in seed dormancy, but whether their mechanism of action relies on a unique dominant signaling pathway or on the overlap of many is still under investigation. Freshly harvested sunflower (Helianthus annuus L.) seeds germinate poorly at temperatures below 10°C, but a short treatment by hydrogen cyanide (HCN) allows their subsequent germination at this temperature, suggesting that HCN might be an important factor in regulation of seed dormancy. Considering the role of reactive oxygen species (ROS) in various physiological processes, I have investigated whether ROS could be involved in HCN-mediated embryo dormancy removal in sunflower embryos. ROS (e.g. hydrogen peroxide) are progressively accumulated in cells of embryonic axes after HCN treatment (Figure 2). Imbibition of embryos in the presence of methylviologen (MV), a ROS generating compound, mimicked the stimulatory effect of HCN on germination, suggesting that it might improve germination through its effect on ROS metabolism and signaling.


Figure 4. In situ localization of ROS production in the embryonic axes of sunflower embryos. A, Embryo showing axe (a) and cotyledon (c; inset frame corresponds to the view shown in B). B, Staining of embryonic axis by toluidine blue (inset frame corresponds to the views shown in confocal). C, Schematic representation of views shown in D to F. c, Cortex; p, pericycle; rc, root cap; qc, quiescent center; lrc, lateral root cap; v, vascular. D to F, Representative fluorescence images of sections of embryonic axes treated with DCFH-DA viewed by confocal laser scanning microscopy. Axes from dormant embryos imbibed for 24 h on water (D), from nondormant embryos imbibed for 24 h on water (E), and from dormant embryos treated for 3 h with cyanide and further imbibed on water (F; total treatment time was 24 h). (Oracz et al. 2009)

Regulation of the transcription of the main genes involved in ROS production and ROS sensing by HCN was investigated using real time RT-PCR, and demonstrated that HCN mainly acts at a post-translational level (Oracz et al. 2009). The crosstalk between HCN, ROS and ethylene in alleviation of sunflower embryo dormancy was discussed, with regards to the role of HCN and ROS in oxidation of specific proteins (Figure 2).


Figure 5. Two-dimensional profiles of protein oxidation in axes of dormant and non-dormant sunflower seeds after 3 h imbibition at 10°C under various conditions. (a) Dormant axes and (b) non-dormant axes imbibed on water; (c) dormant axes and (d) non-dormant axes imbibed in the presence of 1 mM HCN; (e) dormant axes and (f) non-dormant axes imbibed in the presence of 0.1 mM methylviologen. Numbers indicated on the arrows correspond to proteins that have been identified by mass spectrometry (see Tables 3 and 4). Yellow circles, proteins carbonylated in dormant axes but not in non-dormant axes during imbibition on water. Blue circles, proteins specifically carbonylated in the presence of hydrogen cyanide or methylviologen. Green and purple circles, proteins specifically carbonylated in the presence of cyanide or methylviologen, respectively, during imbibition of dormant axes (Oracz et al. 2007a).

Obtained data allow proposing the protein oxidation as a new mechanism of action for HCN and ROS and suggesting that changes in gene expression are not the only mechanism leading to seed dormancy release (Figure 2, 3).


Figure 6. A scheme showing the interactions between HCN (CN in image) and ROS in dormancy alleviation, also integrating data from Oracz et al. (2007, 2008).

Positive regulators of dormancy alleviation are written in bold and negative ones in italic. ETR1, ETR2, Ethylene receptor 1 and 2, respectively; ERS1, ethylene response factor 1; CTR1, constitutive triple response 1; ERF1, ethylene response factor 1; AdoMet, S-adenosylmethionine;ACC, 1-aminocyclopropane 1-carboxylic acid; prot., protein; ox. prot., oxidized protein (Oracz et al. 2009). Moreover, is demonstrated for the first time, that the mechanisms involved in seed dormancy alleviation by hydrogen cyanide unravel the role of reactive oxygen species as key factors of cellular signaling during germination (Figure 3). The complete results are presented and discussed in the following publications Oracz et al., 2007a, 2008, 2009.